The invention relates to an extraordinarily efficient method of inactivating lipid-enveloped viruses such as herpes or retroviruses in biological or biotechnologicalxe2x80x94particularly pharmaceuticalxe2x80x94products, as well as in cell cultures by adding a cyclic lipopeptide or a mixture of lipopeptides or salts or esters thereof at specific concentrations. Lipopeptides were found to have a surprisingly high inactivation potential for lipid-enveloped viruses and in addition, they offer the advantage of an exceedingly low in vivo toxicity, so that the step of removing the inactivating agent from pharmaceutical products or cell cultures can be omitted. The invention is also directed to new antiviral lipopeptides which belong to the surfactins.
At the latest, the AIDS epidemic has brought the realization into the mind of the general public that not only HI viruses but rather, a variety of agents pathogenic for humans may be transmitted, for example, via blood transfusions, pharmaceuticals, transplantations, etc.. In general, it is obligatory today that each pharmaceutical agent prepared from biological material or having come in contact with same is rated as potentially contaminated by microbes or viruses, and that infection-related safety is verified. As a result of the development of molecular-biological methods for manufacturing pharmaceutical agents, the risk of infection by various microbial contaminants has increased further. Animal or human cell lines are frequently used in the biotechnological production of pharmaceuticals. In these cells, in particular, virus infections by endogenous viruses, latent virus infections, or contaminations cannot be excluded completely. The infection-related safety of biotechnological pharmaceuticals, e.g., vaccines, monoclonal antibodies, hormones, or recombinant proteins therefore necessitates removal of any infectious, undesirable particles, which, in principle, may be associated with a considerable loss of research time and means, or productivity. Virus safety of blood and blood products can only be guaranteed by testing and selecting the blood donations, in combination with evaluating and prophylactically using efficient and reliable methods of virus inactivation and elimination.
To inactivate and eliminate viruses from pharmaceutical products, various methods are employed alone or in combination. Chromatographic methods, pH shift, extraction, and fractionation using various organic solvents, salt precipitation, heat treatment, and filtration techniques are employed in case of structurally simple and stable products [Rabenau and Doerr (1990), xe2x80x9cDie Infektionssicherheit biotechnologischer Pharmazeutika aus virologischer Sichtxe2x80x9d, p. 58, GIT VERLAG GmbH, Darmstadt]. Where sensitive or complex biological materials are involved, substances having an antiviral effect are frequently used. Inter alia, the following methods are employed:
the combined use of solvents (e.g., extraction with ether) and synthetic detergents (e.g., Triton X-100) [B. Horowitz et al. (1985), Transfusion 25, 516-522];
the use of xcex2-propiolactone in combination with UV light, as well as methylene blue in combination with photoactivation [W. Stephan (1989) pp. 122-127, in J.-J. Morgenthaler (Ed.); Virus Inactivation in Plasma Products; Curr. Stud. Hematol. Blood Transfus. No. 56, Karger, Basel];
pasteurization of liquid material [T. Nowak (1992), Biologicals 20, 83-85];
heating of lyophilized material [D. Piszkiewicz et al. (1989), pp. 44-54, in J.-J. Morgenthaler (Ed.), Virus Inactivation in Plasma Products., Curr. Stud. Hematol. Blood Transfus. No. 56, Karger, Basel];
irradiation with gamma rays (e.g., cobalt-60) [B. Horowitz et al. (1988), Transfusion 25, 523-527].
The literature describes a number of virus inactivation methods for blood products, particularly human blood plasma. Thus, in U.S. Pat. No. 4,591,505, A. M. Prince discloses an inactivation method for hepatitis B virus wherein the blood products are added with alcohol and either a non-ionic detergent or an ether or a mixture of both as virus-inactivating agent. Polyoxyethylene derivatives or sulfobetains are used as non-ionic detergents.
In U.S. Pat. No. 4,841,023 and in Vox-Sang. 54, 14-20 (1988), S. Karger A G, Basel, B. Horowitz describes the inactivation of lipid-containing viruses in blood products by fatty acids, and in U.S. Pat. No. 4,613,501 by C1-C4 alkyl oleic acid.
In EP 0,050,061 E. Shanbrom discloses a method of reducing undesirable effects such as pyrogenicity, hepatitis infectiousness, and aggregation in biological and pharmaceutical products, particularly blood products as well, using a treatment with non-denaturing amphiphilics such as non-ionic surfactants (e.g., Tween 80).
The inactivation and elimination of viruses from cell cultures is effected using antiviral substances which normally inhibit virus replication.
None of the inactivation methods used up to now can safely inactivate or eliminate all viruses which may occur in biological material. Methods such as pasteurization or heat treatment normally require the use of stabilizers, and in addition, there is the problem of protein denaturation. To date, the use of solvents and synthetic surfactants considered as suitable for inactivating lipid-enveloped viruses could not be assessed as entirely safe due to deviating results in inactivation kinetics or lacking systematic investigations as a result of high toxicity of the substances in cell cultures. Owing to their structure or stability, a variety of biotechnological products cannot be subjected to expensive purification or inactivation using product-damaging or cytotoxic antiviral substances such as solvents.
It was therefore the object of the invention to provide a mild method of inactivating lipid-enveloped viruses in biological or biotechnological products and cell cultures, which method enables to render these products or cell cultures free of viruses in an exceedingly rapid and effective fashion, with no denaturing of products or impairing the cell cultures in their productivity. Also, said method should allow the treatment of heat-labile products and avoid substances having in vivo toxicity.
The object of the invention is accomplished by using cyclic peptides containing xcex2-hydroxyfatty acids and xcex2-aminofatty acids (lipopeptides). These lipopeptides were found to have a surprisingly high inactivation potential for lipid-enveloped viruses, and thus, they are excellent for use in accomplishing the object of the invention. Part of these lipopeptides were found to be substantially more effective compared to synthetic surfactants used in virus inactivation up to now. In addition, they readily undergo biological degradation and have a substantially lower in vivo toxicity than synthetic surfactants. Compared to conventional antiviral substances, the lipopeptides used according to the invention have the advantage of thermal stability and good water-solubility.
From the literature, two [Ile7] and [Leu7] surfactins which belong to the lipopeptides, are known to exhibit moderate anti-HIV-1 activity (H. Itokawa et al., Chem. Pharm. Bull. 42, 604-607 (1994)). In Journal of Antibiotics, Japan XLIII 267-280 (1989), N. Naruse et al. describe pumilacidins as antivirally effective against herpes simplex virus (HSV-1). Neither of these papers indicates the considerable inactivation potential of these substances, allowing extensive inactivation of lipid-enveloped viruses at low concentrations within an exceedingly short time.
Thus, the inactivation method according to the invention is characterized in that the biological or biotechnological products are added with a lipopeptide or a salt or ester thereof, or a mixture of lipopeptides or salts or esters thereof at an overall concentration of 1-100 xcexcM, preferably 1-80 xcexcM, and that the inactivation is performed at room temperature within from 30 min to 2 hours at maximum, with about 99% of the viruses already being inactivated after 30 min. As a result of the exceedingly low in vivo toxicity of the lipopeptides used according to the invention, these inactivating substances may also be allowed to remain in the pharmaceutical products at the above-mentioned concentrations. Following inactivation, the lipopeptides employed may also be removed from the products by reversed phase HPLC on a C18 column, or by adsorption chromatography on a silica gel column.
As the lipopeptides employed according to the invention are thermally stable, the inactivation process may also be performed at elevated temperatures, preferably at 30-60xc2x0 C., depending on the thermal stability of the products to be treated. The inactivation efficiency was found to have a linear temperature dependence. Thus, a temperature increase of about 10xc2x0 C. already results in an increase of the inactivation rate by a factor of about 2.4, so that virus inactivation is possible even within 5-30 min at 30-60xc2x0 C. at and the above-mentioned concentrations. In general, the viruses may also be inactivated at temperatures as low as 0xc2x0 C. Depending on the particular species, however, this may take longer than 2 hours.
According to the invention, the virus inactivation in cell cultures is characterized in that the serum-free culture medium is added with a lipopeptide or a salt or ester thereof, or a mixture of lipopeptides or salts or esters thereof at an overall concentration of 1-65 xcexcM, preferably 1-50 xcexcM. When using a culture medium containing serum up to 5 vol.-%, e.g., FCS, the lipopeptide concentration required for complete inactivation is 10-100 xcexcM, preferably 30-90 xcexcM.
The inactivation process of the invention may be carried out within a broad pH range of 4-9, preferably 5.5-8.
Naturally occurring, chemically synthesized cyclic lipopeptides, as well as those produced and modified by genetic engineering may be used in the inactivation process of the invention.
The cyclic lipopeptides used according to the invention can readily be prepared according to previously described methods well-known to those skilled in the art. The Bacillus subtilis microorganism, among others, forms numerous lipopeptides in vivo which undergo secretion into the surrounding medium at high concentrations, from which they may be isolated.
Predominantly, viruses which can be inactivated by means of the method of the invention are herpes viruses preferably HSV-1, HSV-2, BHV-1, SHV-1, immunodeficiency viruses, preferably HIV-1, HIV-2, SIVagm, the vesicular stomatitis virus (VSV), and the Semliki-Forest virus (SFV). According to the invention, however, other lipid-enveloped viruses may also be inactivated effectively.
According to the invention, lipoheptapeptides of general formula I, 
also referred to as surfactins, the salts, esters or mixtures thereof are preferably used in the inactivation, in which formula I X and Y represent the amino acids Leu, Ile or Val, Z represents the amino acids Val or Ala, and C10-12 represents a linear or branched, saturated alkyl chain. According to the invention, the surfactins of general formula I, with X being Val or Ile, and the esters thereof are new compounds which are also subject of the present invention.
For example, the surfactin mixture produced by the strain Bacillus subtilis ATCC 21332 and the more productive strain Bacillus subtilis OKB 105 was found to be highly suitable in the virus inactivation according to the invention. The surfactin from these Bacillus subtilis strains is a mixture of isoforms, i.e., compounds of general formula I differing in the chain length of the fatty acid, the branching of the fatty acid, and the amino acids X, Y and Z, as illustrated above.
Individual compounds of general formula I, e.g., surfactin isoforms having a fatty acid residue of C14 alkyl or C15 alkyl (i.e., C11 or C12 alkyl in general formula I) which, for example, may be isolated from the surfactin mixture obtained by fermentation of the above-mentioned strains or synthesized by chemical means, likewise exhibit a high inactivation potential. Thus, it has been determined that, e.g., surfactins of general formula I having C15 alkyl as fatty acid residue inactivate vesicular stomatitis virus (VSV) even more rapidly compared to the surfactin mixture. Surfactins having C14 alkyl as fatty acid residue were found to be effective compounds in the inactivation of porcine herpes virus (SHV-1).
Pumilacidins of general formula I in the form of individual components or mixtures, such as described in Journal of Antibiotics, Japan XLIII 267-280 (1989), pp. 267-280, for example, as well as the salts or esters thereof may also be used according to the invention.
In one specific embodiment, compounds of general formula I wherein the amino acids Glu and/or Asp are esterified are employed in the process of the invention. The monoesters of the compounds of general formula I, wherein just one of the above-mentioned amino acids is esterified, exhibit quite specific effects. Thus, the C14 alkyl monoester of general formula I, for example, was found to inactivate the porcine herpes virus within 20 minutes at a concentration of 40 xcexcM by a factor of  less than 104. The C15 alkyl monoesters of general formula I were also capable of inactivating the Semliki-Forest virus (SFV) within 20 minutes at a concentration of 40 xcexcM by a factor of  greater than 104. The inactivation of SFV using C14 or C15 alkyl monoesters is exemplified in Table 1 below:
In the meaning of the invention, biological products represent products isolated from mammals, such as blood products, products isolated from blood, such as vaccines and plasma derivatives. Biotechnological pharmaceutical products are understood to be active substances produced by biotechnological means, such as human proteins (hGH, TNF, t-PA, EPO), or coagulation factors (e.g., factor VIII); however, the invention is not limited to the above-mentioned products from cell cultures.
With reference to the embodiments, the invention will be illustrated in more detail below.